Lipid nanoparticle formulations and methods of synthesis thereof

Abstract

Stabilized formulations of lipids and nucleic acids, including lipid nanoparticle formulations which encapsulate nucleic acids are disclosed herein. Methods of using the formulations are also provided.

1 . A method of producing a lipid nanoparticle composition, the method comprising:
 a) mixing a lipid solution comprising 0.25 mol % to 0.75 mol % of a first PEG-lipid, an ionizable lipid, a phospholipid, and a structural lipid with an aqueous buffer to thereby form a precursor lipid nanoparticle; and   b) adding a lipid nanoparticle modifier comprising 1.5 mol % to 2.5 mol % of a second PEG-lipid to the precursor lipid nanoparticle thereby forming a modified lipid nanoparticle.   
     
     
         2 . The method of  claim 1 , further comprising mixing an mRNA with the lipid solution and the aqueous buffer to thereby form the precursor lipid nanoparticle, which is a precursor nucleic acid lipid nanoparticle. 
     
     
         3 . The method of  claim 1 , further comprising mixing an mRNA with the precursor lipid nanoparticle to thereby form an mRNA-lipid nanoparticle. 
     
     
         4 . The method of  claim 1 , wherein the lipid nanoparticle modifier is added in two separate stages and the total amount of the second PEG-lipid added is 1.5 mol % to 2.5 mol %. 
     
     
         5 . The method of  claim 1 , wherein the lipid nanoparticle modifier is added in three, four, or five separate stages and the total amount of the second PEG-lipid added is 1.5 mol % to 2.5 mol %. 
     
     
         6 . The method of  claim 1 , wherein the lipid solution comprises 0.5 mol % of the first PEG-lipid. 
     
     
         7 . The method of  claim 6 , wherein the lipid nanoparticle modifier comprises 2.0 mol % of the second PEG-lipid. 
     
     
         8 . The method of  claim 1 , wherein the modified lipid nanoparticle comprises a total of 2.5 mol % of the first PEG-lipid and the second PEG-lipid. 
     
     
         9 . (canceled) 
     
     
         10 . The method of  claim 1 , wherein the first PEG-lipid is PEG-DMG or 134-hydroxy-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78,81,84,87,90,93,96,99,102,105,108,111,114,117,120,123,126,129,132-tetratetracontaoxatetratriacontahectyl stearate. 
     
     
         11 . The method of  claim 10 , wherein the second PEG-lipid is PEG-DMG or 134-hydroxy-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78,81,84,87,90,93,96,99,102,105,108,111,114,117,120,123,126,129,132-tetratetracontaoxatetratriacontahectyl stearate. 
     
     
         12 . The method of  claim 1 , wherein the ionizable lipid is compound I ((heptadecan-9-yl 8 ((2 hydroxyethyl)(6 oxo 6-(undecyloxy) hexyl)amino) octanoate)), compound II (heptadecan-9-yl 8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate), compound III (heptadecan-9-yl 8-((3-((2-(methylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)propyl)(8-oxo-8-(undecan-3-yloxy)octyl)amino)octanoate), or compound IV (3-butylheptyl 8-((8-(heptadecan-9-yloxy)-8-oxooctyl) (2-hydroxyethyl)amino)octanoate). 
     
     
         13 . The method of  claim 12 , wherein the phospholipid is distearoylphosphatidylcholine (DSPC). 
     
     
         14 . The method of  claim 13 , wherein the structural lipid is a sterol. 
     
     
         15 . (canceled) 
     
     
         16 . The method of  claim 1 , wherein the lipid solution comprises ethanol. 
     
     
         17 . The method of  claim 1 , wherein the mixing in (a) comprises turbulent mixing (“T-mix”), vortex mixing (“V-mix”), microfluidic mixing, or a combination thereof. 
     
     
         18 . (canceled) 
     
     
         19 . A composition comprising the mRNA-lipid nanoparticle of  claim 3 . 
     
     
         20 - 21 . (canceled) 
     
     
         22 . The composition of  claim 19 , wherein the composition is stable for at least six months at a temperature of 2° C. to about 6° C. 
     
     
         23 . The composition of  claim 19 , wherein the composition is stable for at least six months at room temperature. 
     
     
         24 . The composition of  claim 19 , wherein the mRNA-lipid nanoparticle comprises about 2-3 mol % PEG-modified lipid, 5-25 mol % neutral lipid, 25-55 mol % sterol, and 20-60% ionizable amino lipid. 
     
     
         25 .- 40 . (canceled) 
     
     
         41 . A composition comprising a lipid nanoparticle (LNP) and a nucleic acid, wherein the LNP comprises 2.5 mol % 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 11 mol % 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 38.5 mol % cholesterol, and 48 mol % Compound I: 
       
         
           
           
               
               
           
         
       
       (heptadecan-9-yl 8 ((2 hydroxyethyl)(6 oxo 6-(undecyloxy) hexyl)amino) octanoate), and
 wherein 0.25 to 0.5 mol % of the PEG-DMG is in the core of the LNP. 
 
     
     
         42 .- 44 . (canceled)